How consistent are we? Interlaboratory comparison study in fathead minnows using the model estrogen 17α-ethinylestradiol to develop recommendations for environmental transcriptomics.

Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of the present study was to determine the congruence of transcript data across 6 independent laboratories. Male fathead minnows were exposed to a measured concentration of 15.8 ng/L 17α-ethinylestradiol (EE2) for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same fathead minnow microarray. Each laboratory was free to apply bioinformatics pipelines of its choice. There were 12 491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was approximately 50%, which improved to approximately 59.0% using a standardized analysis pipeline. The dynamic range of fold change estimates was variable between laboratories, but ranking transcripts by their relative fold difference resulted in a positive relationship for comparisons between any 2 laboratories (mean R2 > 0.9, p < 0.001). Ten estrogen-responsive genes encompassing a fold change range from dramatic (>20-fold; e.g., vitellogenin) to subtle (∼2-fold; i.e., block of proliferation 1) were identified as differentially expressed, suggesting that laboratories can consistently identify transcripts that are known a priori to be perturbed by a chemical stressor. Thus, attention should turn toward identifying core transcriptional networks using focused arrays for specific chemicals. In addition, agreed-on bioinformatics pipelines and the ranking of genes based on fold change (as opposed to p value) should be considered in environmental risk assessment. These recommendations are expected to improve comparisons across laboratories and advance the use of omics in regulations. Environ Toxicol Chem 2017;36:2593-2601. © 2017 SETAC.

Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis.

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

Defining the role of omics in assessing ecosystem health: Perspectives from the Canadian environmental monitoring program.

Scientific reviews and studies continue to describe omics technologies as the next generation of tools for environmental monitoring, while cautioning that there are limitations and obstacles to overcome. However, omics has not yet transitioned into national environmental monitoring programs designed to assess ecosystem health. Using the example of the Canadian Environmental Effects Monitoring (EEM) program, the authors describe the steps that would be required for omics technologies to be included in such an established program. These steps include baseline collection of omics endpoints across different species and sites to generate a range of what is biologically normal within a particular ecosystem. Natural individual variability in the omes is not adequately characterized and is often not measured in the field, but is a key component to an environmental monitoring program, to determine the critical effect size or action threshold for management. Omics endpoints must develop a level of standardization, consistency, and rigor that will allow interpretation of the relevance of changes across broader scales. To date, population-level consequences of routinely measured endpoints such as reduced gonad size or intersex in fish is not entirely clear, and the significance of genome-wide molecular, proteome, or metabolic changes on organism or population health is further removed from the levels of ecological change traditionally managed. The present review is not intended to dismiss the idea that omics will play a future role in large-scale environmental monitoring studies, but rather outlines the necessary actions for its inclusion in regulatory monitoring programs focused on assessing ecosystem health.

Using molecular biomarkers and traditional morphometric measurements to assess the health of slimy sculpin (Cottus cognatus) from streams with elevated selenium in North-Eastern British Columbia.

Canadian fish-based environmental effects monitoring programs use individual and population-level endpoints to assess aquatic health. Impacts of coal mining and selenium (Se) exposure were assessed in slimy sculpin (Cottus cognatus) from reference streams located both inside and outside of a coal zone, and from 1 stream with a history of coal mining, using traditional environmental effects monitoring endpoints. In addition, physical characteristics of the streams and benthic macro-invertebrate communities were assessed. To determine whether the assessment of effects could be improved by including molecular markers, real-time polymerase chain reaction assays were optimized for genes associated with reproduction (vtg, esr1, star, cyp19a1, and gys2), and oxidative and cellular stress (sod1, gpx, gsr, cat, and hsp 90). Water Se levels exceeded guidelines in the stream with historical mining (4 µg/L), but benthic macroinvertebrates did not exceed dietary thresholds (2-3 µg/g dry wt). Whole-body Se levels were above British Columbia's tissue guideline in fish from all streams, but only above the draft US Environmental Protection Agency (USEPA) criterion (7.91 µg/g dry wt) at the reference stream inside the coal zone. Some markers of cellular and oxidative stress were elevated in fish liver at the exposed site (sod1, gpx), but some were lower (cat, sod1, gpx, gsr, hsp90) in the gonads of fish inside the coal zone. Some of the differences in gene expression levels between the reference and impacted sites were sex dependent. Based on benthic macroinvertebrate assessments, the authors hypothesize that traditional and molecular differences in slimy sculpin at impacted sites may be driven by food availability rather than Se exposure. The present study is the first to adapt molecular endpoints in the slimy sculpin for aquatic health assessments.